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REPLI-g Advanced DNA Single Cell Kit

For whole genome amplification (WGA) from single eukaryotic cells, limited samples or purified genomic DNA

Features

  • WGA with complete genome coverage from single eukaryotic cells or 1 ng genomic DNA 
  • Simple 3-step protocol requires only 2 hours and 20 minutes, start to finish
  • Consistent yields of up to 25–35 µg DNA from 1 eukaryotic cell
  • Includes cryo-protect reagent to minimize DNA damage during single cell capture, storage or shipping
REPLI-g Advanced DNA Single Cell Kit (24)

Cat. No. / ID: 150363

Buffers, enzymes and single cell storage solution for 24 WGA reactions
kitreagent
REPLI-g Advanced DNA Single Cell Kit
REPLI-g Single Cell Cryo-Protect Reagent
reactions
24
96

Product Details


DNA sequence analysis and genotyping of biological samples using next-generation sequencing (NGS) platforms is often limited by the amount of available sample. The REPLI-g Advanced DNA Single Cell Kit has been specifically optimized for use in sensitive microarray and NGS applications, providing high uniformity, with minimal allelic dropout, when starting with single cells or purified genomic DNA. Buffers and reagents undergo a unique, controlled decontamination procedure to minimize contaminating DNA and ensure highly reliable results. Accurate amplification of genomes with negligible sequence bias and minimal genomic dropouts is achieved with innovative Multiple Displacement Amplification (MDA) technology.

Performance

Genotyping and DNA sequence analysis of biological samples can be limited by a small amount of available sample. The REPLI-g Advanced DNA Single Cell Kit allows uniform amplification of whole genomic DNA from limited sample amounts and enables a greater variety and number of analyses to be performed. The average product length of amplified DNA is typically more than 10 kb, with a range between 2 kb and 100 kb, enabling all downstream applications such as complex genetic analysis, including long-range copy number variations, to be performed (1). DNA amplified with the REPLI-g Advanced DNA Single Cell Kit is highly suited for next-generation sequencing (NGS), array CGH genotyping applications or qPCR analysis.

Typical DNA yields from REPLI-g Advanced DNA Single Cell Kit amplifications are approximately 25–35 µg per 50 µl reaction. Depending on the quality of the input cell and its DNA, the resulting amount of amplified DNA may be less (fragmented or damaged gDNA should not be used). For best amplification results, we recommend collecting or storing cells in REPLI-g Advanced sc Storage Buffer (included with kit or available separately).

Principle

The REPLI-g Advanced DNA Single Cell Kit contains an optimized Phi 29 polymerase formulation, as well as buffers and reagents, for whole genome amplification (WGA) from single eukaryotic cells, very small amounts of sample or purified genomic DNA using Multiple Displacement Amplification (MDA). Phi 29 polymerase, a phage-derived enzyme, is a DNA polymerase with 3'→5' exonuclease activity (proofreading activity) that delivers up to 1000-fold higher fidelity compared to Taq DNA polymerase. Supported by the unique, optimized REPLI-g Advanced DNA Single Cell buffer system, Phi 29 polymerase easily solves secondary structures such as hairpin loops, thereby preventing slipping, stoppage and dissociation of the polymerase during amplification. The REPLI-g Advanced DNA Single Cell Kit is an improved version of the REPLI-g Single Cell Kit and includes an optimized reaction chemistry, a new single cell storage buffer and an improved protocol to increase the uniformity of amplification and reduce potential amplification bias. In addition, the MDA reaction time has been shortened to 2 hours.

Procedure

The REPLI-g Advanced DNA Single Cell Kit provides highly uniform amplification across the entire genome, with negligible sequence bias. The method is based on Multiple Displacement Amplification (MDA) technology, which carries out isothermal genome amplification utilizing a uniquely processive DNA polymerase capable of replicating up to 100 kb without dissociating from the genomic DNA template. In contrast to PCR-based methods, Phi 29 polymerase has a 3'–5' exonuclease proofreading activity to maintain 1000-fold higher fidelity than Taq Polymerase during replication. MDA technology is used in the presence of exonuclease-resistant primers to achieve high yields of DNA product from all kinds of eukaryotic tissues.

Genetic analyses often require substantial amounts of genomic DNA. Whole genome amplification solves the problem of low DNA quantity when starting with 1–1000 cells. The REPLI-g Advanced DNA Single Cell Kit overcomes these limitations by using a simple and reliable method to achieve accurate genome amplification. In addition, all components provided with the REPLI-g Advanced DNA Single Cell kit are exposed to UV radiation in a standardized procedure to minimize contaminating DNA. Reaction setup takes only 20 minutes and 3 steps, making the REPLI-g Advanced DNA Single Cell Kit protocol easy and reliable.

In the first step of the procedure, the cell sample is lysed under isothermal alkaline conditions and the cell is denatured. Cell lysis at room temperature reduces the number of possible DNA breaks and therefore improves uniformity of whole genome amplification. After denaturation has been stopped by the addition of neutralization buffer, a master mix containing buffer and DNA polymerase is added. The isothermal amplification reaction proceeds for 2 hours at 30°C. Because of the shortened MDA reaction time, the REPLI-g Advanced DNA Single Cell Kit enables single cell collection, WGA and even downstream analysis to be performed in a single day.

Applications

Downstream applications and instrumentation
Application Instrumentation
Whole genome sequencing Next-generation sequencing platforms*
Exome sequencing
SNP genotyping arrays Array platforms*
Array CGH
qPCR/PCR technologies Real-time PCR/PCR cyclers*
Sanger sequencing Capillary sequencers*
Pyrosequencing PyroMark (QIAGEN)
* Available from various suppliers.


Learn more about our single cell products by visiting our Single Cell Resource.


References

1. Hosono, S. et al. (2003) Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13, 954.

Supporting data and figures

Resources

Supplementary Protocols (16)
Kit Handbooks (1)

FAQ

Are there important considerations for plasma generation and urine handling?

It is strongly advised to follow the recommendations for preparing sample material provided in the corresponding Protocol Sheet to ensure reliable results.

Plasma: It is recommended to perform plasma separation immediately after blood collection when using EDTA or citrate as anticoagulant to prevent the release of genomic DNA into the plasma fraction.

Urine: Because circulating cell-free DNA in non-stabilized urine samples is rapidly degraded after sample collection due to high nuclease activity, eluates may contain no DNA or exhibit low DNA concentration. Therefore, it is recommended to stabilize urine samples. Even when using stabilized urine, it is recommended to perform a centrifugation step immediately after stabilization to prevent the release of genomic DNA from cells. Alternatively, non-stabilized urine samples can be processed immediately after collection and centrifugation using ATL-pretreatment and automated DNA extraction as described in the corresponding Protocol Sheet.

FAQ ID - 3699
Can the Repli-g Advanced DNA Single Cell Kit also be used for bacterial cells?
The Repli-G Advanced DNA Single Cell Kit has been developed with eukaryotic cells and this works perfectly.
However, bacterial cells can be more difficult to lyse. Therefore, it might be necessary to incubate the bacterial cells for 10 min at 65°C rather than at room temperature in the lysis buffer D2, to make the lysis more stringent. All succeeding steps should be done according to the standard protocol. The most critical factor to get this protocol to work is the quality of the bacterial DNA after lysis. It is crucial to start with fresh, top quality material.
Our customers already gave us some feedback that the DNA of gram+ as well as gram- bacteria was successfully amplified with the Repli-g Advanced DNA Single Cell Kit using the more stringent lysis temperature.
The Repli-g Advanced DNA Single Cell Kit is a perfect choice especially for small amounts of bacterial starting material because the reagents are decontaminated for any residual DNA, reducing possible artefacts.
FAQ ID - 143079
Can the Repli-g Advanced DNA Single Cell Kit also be used for bacterial cells?
The Repli-g Advanced DNA Single Cell Kit has been developed with eukaryotic cells and this works perfectly. 
However, bacterial cells can be more difficult to lyse. Therefore, it might be necessary to incubate the bacterial cells for 10 min at 65°C rather than at room temperature in the lysis buffer D2, to make the lysis more stringent. All succeeding steps should be done according to the standard protocol. The most critical factor to get this protocol to work is the quality of the bacterial DNA after lysis. It is crucial to start with fresh, top quality material. 
Our customers already gave us some feedback that the DNA of  gram+ as well as gram- bacteria was successfully amplified with the Repli-g Advanced DNA Single Cell Kit using the more stringent lysis temperature. 
The Repli-G Advanced DNA Single Cell Kit is a perfect choice especially for small amounts of bacterial starting material because the reagents are decontaminated for any residual DNA, reducing possible artefacts.
FAQ-ID -143711